This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
CHAPTER 9 - MOLECULAR METHODS
Sections of Whole Mount in situ Hybridization
(Source: R. BreMiller)
1. Use embryos in which alkaline phosphatase
has been employed as a detection enzyme.
For the best results it is important to
overstain heavily the whole-mount
2. Postfix embryos in 4% paraformaldehyde in
PBS. The tissue can be stored in this
solution for an extended period of time in
the dark at 4°C.
3. Wash embryos in dH2O and
dehydrate quickly through a graded series of
methanol (50%, 70%, 95% and twice 100%
2 to 3 min each).
4. Replace the methanol with propylene oxide,
two changes for 5 min
5. Replace with a 1:1 mixture of Epon and
propylene oxide for 30
6. Replace with a 3:1 mixture of
Epon:propylene oxide for 2 to 3
7. Transfer tissue to pure Epon for 4
8. Embed in fresh Epon. Polymerize overnight
in a 60°C
9. Cut sections 5 to 10 µm thick with a
glass knife. Dry down on a drop of water.
Cover slip in Permount. Alternatively,
cover slip in Epon and polymerize at 60°C.
1. Wash stained embryos in PBS, two changes,
2 to 3 min each.
2. Wash in dH2O.
3. Embed embryos in agar-sucrose as described
in Agar Mounting, Chapter 4.
Cryoprotect in 30% sucrose and cut 14 to
16 µm sections in a -20°C cryostat. Pick up sections
on subbed slides and dry at room
temperature 2 to 3
4. Wash sections in dH2O 2 to
3 min and dehydrate through graded
alcohols. Clear in xylene and mount in
NOTES: Aqueous solutions of
0.2% methyl green or 1%
neutral red can be used as
a counterstain for the
frozen sections. Immerse
slides briefly (30 to 90
sec) in the staining
solution, wash in dH2O, and
dehydrate as above.
The Zebrafish Book